Protease variants and polynucleotides encoding same

ABSTRACT

The present invention relates to protease variants and methods for obtaining protease variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2014/066194 filed Jul. 28, 2014, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 13178320.1 filedJul. 29, 2013. The content of each application is fully incorporatedherein by reference.

REFERENCE TO A JOINT RESEARCH AGREEMENT

The inventions claimed in the present application were made under ajoint research agreement between Henkel AG & Co. KGaA and Novozymes A/S.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to novel protease variants exhibitingalterations relative to the parent subtilase in one or more propertiesincluding: wash performance, detergent stability and/or storagestability. The variants of the invention are suitable for use in e.g.cleaning or detergent compositions, such as laundry detergentcompositions and dish wash compositions, including automatic dish washcompositions. The present invention also relates to isolated DNAsequences encoding the variants, expression vectors, host cells, andmethods for producing and using the variants of the invention. Further,the present invention relates to cleaning and detergent compositionscomprising the variants of the invention.

Description of the Related Art

Enzymes have been used within the detergent industry as part of washingformulations for many decades. Proteases are from a commercialperspective the most relevant enzyme in such formulations, but otherenzymes including lipases, amylases, cellulases, hemicellulases ormixtures of enzymes are also often used. To improve the cost and/or theperformance of proteases there is an ongoing search for proteases withaltered properties, such as increased activity at low temperatures,increased stability, increased specific activity at a given pH, alteredCa²⁺ dependency, increased stability in the presence of other detergentingredients (e.g. bleach, surfactants etc.) etc. One family ofproteases, which are often used in detergents, are the subtilases. Thisfamily has previously been further grouped into 6 different sub-groupsby Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523. Oneof these sub-groups is the Subtilisin family which includes subtilasessuch as BPN′, subtilisin 309 (SAVINASE®, Novozymes A/S), subtilisinCarlsberg (ALCALASE®, Novozymes A/S), subtilisin S41 (a subtilase fromthe psychrophilic Antarctic Bacillus TA41, Davail S et al. 1994, TheJournal of Biological Chemistry, 269(26), 99. 17448-17453) andsubtilisin S39 (a subtilase from the psychrophilic Antarctic BacillusTA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp.1271-1279). TY145 is a subtilase from Bacillus sp. TY145, NCIMB 40339,which were first described in WO 92/17577 (Novozymes NS) and in thelater application WO2004/067737 (Novozymes NS) disclosing thethree-dimensional structure and the use of protein engineering to alterfunctionality of a TY-145 subtilase.

SUMMARY OF THE INVENTION

The present invention relates to protease variants, comprising analteration at one or more (e.g., several) positions corresponding topositions 121, 124, 137 and 162 of SEQ ID NO: 3, wherein the varianthave protease activity and wherein the variants has an amino acidsequence which is at least 70% identical to the mature polypeptide ofSEQ ID NO: 2 or to SEQ ID NO: 3.

The present invention relates to a method for obtaining a proteasevariant, comprising introducing into a parent subtilase a substitutionat one or more positions in the hydrophobic cluster around position 137of SEQ ID NO: 3 corresponding to positions 121, 124, 137 and 162 of SEQID NO: 3, wherein the variant has an amino acid sequence which is atleast 70% identical to SEQ ID NO: 3; and recovering the variant. Thepresent invention also relates to isolated polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing the variants.

Overview of Sequences Listing

-   SEQ ID NO: 1=is the DNA sequence of TY-145 protease isolated from    Bacillus sp.-   SEQ ID NO: 2=is the amino acid sequence as deduced from SEQ ID NO:    1.-   SEQ ID NO: 3=is the amino acid sequence of the mature TY145.

Definitions

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996,237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999,264, 610-650; respectively. The term “subtilases” refer to a sub-groupof serine protease according to Siezen et al., Protein Engng. 4 (1991)719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serineproteases or serine peptidases is a subgroup of proteases characterisedby having a serine in the active site, which forms a covalent adductwith the substrate. Further the subtilases (and the serine proteases)are characterised by having two active site amino acid residues apartfrom the serine, namely a histidine and an aspartic acid residue. Thesubtilases may be divided into 6 sub-divisions, i.e. the Subtilisinfamily, the Thermitase family, the Proteinase K family, the Lantibioticpeptidase family, the Kexin family and the Pyrolysin family.

The term “protease activity” means a proteolytic activity (EC 3.4).Proteases of the invention are endopeptidases (EC 3.4.21). There areseveral protease activity types: The three main activity types are:trypsin-like where there is cleavage of amide substrates following Argor Lys at P1, chymotrypsin-like where cleavage occurs following one ofthe hydrophobic amino acids at P1, and elastase-like with cleavagefollowing an Ala at P1. For purposes of the present invention, proteaseactivity is determined according to the procedure described in“Materials and Methods” below. The subtilase variants of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the protease activity of the mature polypeptide with SEQ ID NO:3.

The term “parent” or protease parent means a protease to which analteration is made to produce the enzyme variants of the presentinvention. Thus the parent is a protease having the identical amino acidsequence of said variant but not having the alterations at one or moreof said specified positions. It will be understood, that in the presentcontext the expression “having identical amino acid sequence” relates to100% sequence identity. The parent may be a naturally occurring(wild-type) polypeptide or a variant thereof. In a particular embodimentthe parent is a protease with at least 70%, at least 75%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to a polypeptide with SEQ ID NO: 3.

The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (or one or several) positions compared to itsparent which is a protease having the identical amino acid sequence ofsaid variant but not having the alterations at one or more of saidspecified positions. A substitution means a replacement of an amino acidoccupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding amino acids e.g. 1 to 10 amino acids, preferably 1-3 amino acidsadjacent to an amino acid occupying a position.

The term “isolated variant” means a variant that is modified by the handof man. In one aspect, the variant is at least 1% pure, e.g., at least5% pure, at least 10% pure, at least 20% pure, at least 40% pure, atleast 60% pure, at least 80% pure, and at least 90% pure, as determinedby SDS-PAGE.

The term “isolated polynucleotide” means a polynucleotide that ismodified by the hand of man. In one aspect, the isolated polynucleotideis at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least20% pure, at least 40% pure, at least 60% pure, at least 80% pure, atleast 90% pure, and at least 95% pure, as determined by agaroseelectrophoresis. The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “wild-type protease” means a protease expressed by a naturallyoccurring organism, such as a bacterium, archaea, yeast, fungus, plantor animal found in nature. An example of a wild-type protease is TY-145i.e. the mature polypeptide of SEQ ID NO: 2 i.e. amino acids 1 to 311 orthe mature polypeptide with SEQ ID NO: 3.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In one aspect, the mature polypeptide correspondsto the amino acid sequence with SEQ ID NO: 3.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having protease activity. In oneaspect, the mature polypeptide coding sequence is nucleotides 331 to1263 of SEQ ID NO: 1 based on the SignalP (Nielsen et al., 1997, ProteinEngineering 10: 1-6)] that predicts nucleotides 1 to 81 of SEQ ID NO: 1is the signal peptide.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aprokaryotic or eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of its polypeptide product. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic. The termnucleic acid construct is synonymous with the term “expression cassette”when the nucleic acid construct contains the control sequences requiredfor expression of a coding sequence of the present invention.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence.

The term “control sequences” means all components necessary for theexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native or foreign to thepolynucleotide encoding the variant or native or foreign to each other.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

The term “expression” includes any step involved in the production ofthe variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to additional nucleotides that provide for its expression.

The term “transcription promoter” is used for a promoter which is aregion of DNA that facilitates the transcription of a particular gene.Transcription promoters are typically located near the genes theyregulate, on the same strand and upstream (towards the 5′ region of thesense strand).

The term “transcription terminator” is used for a section of the geneticsequence that marks the end of gene or operon on genomic DNA fortranscription.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, and the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and either 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “improved property” means a characteristic associated with avariant that is improved compared to the parent or compared to aprotease with SEQ ID NO: 3, or compared to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. Such improvedproperties include, but are not limited to, wash performance, proteaseactivity, thermal activity profile, thermostability, pH activityprofile, pH stability, substrate/cofactor specificity, improved surfaceproperties, substrate specificity, product specificity, increasedstability, improved stability under storage conditions, and chemicalstability.

The term “improved protease activity” is defined herein as an alteredprotease activity (as defined above) of a protease variant displaying analteration of the activity relative (or compared) to the activity of theparent subtilase, or compared to a protease with SEQ ID NO: 3, orrelative to a protease having the identical amino acid sequence of saidvariant but not having the alterations at one or more of said specifiedpositions, by increased protein conversion.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process and reflects the stability of theprotease variant according to the invention as a function of time e.g.how much activity is retained when the protease variant is kept insolution in particular in a detergent solution. The stability isinfluenced by many factors e.g. pH, temperature, detergent compositione.g. amount of builder, surfactants etc. The protease stability may bemeasured using the assay described in example 2. The term “improvedstability” or “increased stability” is defined herein as a variantprotease displaying an increased stability in solutions, relative to thestability of the parent protease, relative to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions or relative toSEQ ID NO: 3. The terms “improved stability” and “increased stability”includes “improved chemical stability”, “detergent stability” or“improved detergent stability.

The term “improved chemical stability” is defined herein as a variantenzyme displaying retention of enzymatic activity after a period ofincubation in the presence of a chemical or chemicals, either naturallyoccurring or synthetic, which reduces the enzymatic activity of theparent enzyme. Improved chemical stability may also result in variantsbeing more able to catalyze a reaction in the presence of suchchemicals. In a particular aspect of the invention the improved chemicalstability is an improved stability in a detergent, in particular in aliquid detergent. The term “detergent stability” or “improved detergentstability is in particular an improved stability of the proteaseactivity when a protease variant of the present invention is mixed intoa liquid detergent formulation and then stored at temperatures between15 and 50° C. In the present invention liquid detergents are particularuseful as liquid laundry detergents.

The term “improved thermal activity” means a variant displaying analtered temperature-dependent activity profile at a specific temperaturerelative to the temperature-dependent activity profile of the parent orrelative to a protease with SEQ ID NO: 3. The thermal activity valueprovides a measure of the variant's efficiency in enhancing catalysis ofa hydrolysis reaction over a range of temperatures. A more thermo activevariant will lead to an increase in enhancing the rate of hydrolysis ofa substrate by an enzyme composition thereby decreasing the timerequired and/or decreasing the enzyme concentration required foractivity. Alternatively, a variant with a reduced thermal activity willenhance an enzymatic reaction at a temperature lower than thetemperature optimum of the parent defined by the temperature-dependentactivity profile of the parent.

The term “improved wash performance” is defined herein as a proteasevariant according to the invention displaying an improved washperformance relative to the wash performance of the parent protease,relative to a protease with SEQ ID NO: 3 or relative to a proteasehaving the identical amino acid sequence of said variant but not havingthe alterations at one or more of said specified positions e.g. byincreased stain removal. The term “wash performance” includes washperformance in laundry but also e.g. in dish wash. The wash performancemay be quantified as described under the definition of “washperformance” herein. The terms “cleaning compositions” and “cleaningformulations,” refer to compositions that find use in the removal ofundesired compounds from items to be cleaned, such as fabric, carpets,dishware including glassware, contact lenses, hard surfaces such astiles, zincs, floors, and table surfaces, hair (shampoos), skin (soapsand creams), teeth (mouthwashes, toothpastes), etc. The termsencompasses any materials/compounds selected for the particular type ofcleaning composition desired and the form of the product (e.g., liquid,gel, granule, or spray compositions), as long as the composition iscompatible with the protease variants according to the invention andother enzyme(s) used in the composition. The specific selection ofcleaning composition materials is readily made by considering thesurface, item or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use. These terms furtherrefer to any composition that is suited for cleaning, bleaching,disinfecting, and/or sterilizing any object and/or surface. It isintended that the terms include, but are not limited to detergentcomposition (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

The term “detergent composition”, includes unless otherwise indicated,granular or powder-form all-purpose or heavy-duty washing agents,especially cleaning detergents; liquid, gel or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL) types;liquid fine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, cleaningbars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos,bathroom cleaners; hair shampoos and hair-rinses; shower gels, foambaths; metal cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types.

The terms “detergent composition” and “detergent formulation” are usedin reference to mixtures which are intended for use in a wash medium forthe cleaning of soiled objects. In some embodiments, the term is used inreference to laundering fabrics and/or garments (e.g., “laundrydetergents”). In alternative embodiments, the term refers to otherdetergents, such as those used to clean dishes, cutlery, etc. (e.g.,“dishwashing detergents”). It is not intended that the present inventionbe limited to any particular detergent formulation or composition. It isintended that in addition to the variants according to the invention,the term encompasses detergents that contains, e.g., surfactants,builders, chelators or chelating agents, bleach system or bleachcomponents, polymers, fabric conditioners, foam boosters, sudssuppressors, dyes, perfume, tannish inhibitors, optical brighteners,bactericides, fungicides, soil suspending agents, anti corrosion agents,enzyme inhibitors or stabilizers, enzyme activators, transferase(s),hydrolytic enzymes, oxido reductases, bluing agents and fluorescentdyes, antioxidants, and solubilizers.

The term “fabric” encompasses any textile material. Thus, it is intendedthat the term encompass garments, as well as fabrics, yarns, fibers,non-woven materials, natural materials, synthetic materials, and anyother textile material.

The term “textile” refers to woven fabrics, as well as staple fibers andfilaments suitable for conversion to or use as yarns, woven, knit, andnon-woven fabrics. The term encompasses yarns made from natural, as wellas synthetic (e.g., manufactured) fibers. The term, “textile materials”is a general term for fibers, yarn intermediates, yarn, fabrics, andproducts made from fabrics (e.g., garments and other articles).

The term “non-fabric detergent compositions” include non-textile surfacedetergent compositions, including but not limited to dishwashingdetergent compositions, oral detergent compositions, denture detergentcompositions, and personal cleansing compositions.

The term “effective amount of enzyme” refers to the quantity of enzymenecessary to achieve the enzymatic activity required in the specificapplication, e.g., in a defined detergent composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular enzyme used, thecleaning application, the specific composition of the detergentcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like. The term “effective amount” of aprotease variant refers to the quantity of protease variant describedhereinbefore that achieves a desired level of enzymatic activity, e.g.,in a defined detergent composition.

The term “water hardness” or “degree of hardness” or “dH” or “° dH” asused herein refers to German degrees of hardness. One degree is definedas 10 milligrams of calcium oxide per liter of water.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,detergent concentration, type of detergent and water hardness, actuallyused in households in a detergent market segment.

The term “adjunct materials” means any liquid, solid or gaseous materialselected for the particular type of detergent composition desired andthe form of the product (e.g., liquid, granule, powder, bar, paste,spray, tablet, gel, or foam composition), which materials are alsopreferably compatible with the protease variant enzyme used in thecomposition. In some embodiments, granular compositions are in “compact”form, while in other embodiments, the liquid compositions are in a“concentrated” form.

The term “stain removing enzyme” as used herein, describes an enzymethat aids the removal of a stain or soil from a fabric or a hardsurface. Stain removing enzymes act on specific substrates, e.g.,protease on protein, amylase on starch, lipase and cutinase on lipids(fats and oils), pectinase on pectin and hemicellulases onhemicellulose. Stains are often depositions of complex mixtures ofdifferent components which either results in a local discolouration ofthe material by itself or which leaves a sticky surface on the objectwhich may attract soils dissolved in the washing liquor therebyresulting in discolouration of the stained area. When an enzyme acts onits specific substrate present in a stain the enzyme degrades orpartially degrades its substrate thereby aiding the removal of soils andstain components associated with the substrate during the washingprocess. For example, when a protease acts on a grass stain it degradesthe protein components in the grass and allows the green/brown colour tobe released during washing.

The term “reduced amount” means in this context that the amount of thecomponent is smaller than the amount which would be used in a referenceprocess under otherwise the same conditions. In a preferred embodimentthe amount is reduced by, e.g., at least 5%, such as at least 10%, atleast 15%, at least 20% or as otherwise herein described.

The term “low detergent concentration” system includes detergents whereless than about 800 ppm of detergent components is present in the washwater. Asian, e.g., Japanese detergents are typically considered lowdetergent concentration systems.

The term “medium detergent concentration” system includes detergentswherein between about 800 ppm and about 2000 ppm of detergent componentsis present in the wash water. North American detergents are generallyconsidered to be medium detergent concentration systems.

The term “high detergent concentration” system includes detergentswherein greater than about 2000 ppm of detergent components is presentin the wash water. European detergents are generally considered to behigh detergent concentration systems.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 3 is used to determine the corresponding amino acidresidue in another protease. The amino acid sequence of another proteaseis aligned with the mature polypeptide disclosed in SEQ ID NO: 3, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the mature polypeptide disclosed in SEQ ID NO:3 is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 5.0.0 or later. The parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 3 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP super families of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letters amino acid abbreviations are employed.Amino acid positions are indicated with #₁, #₂, etc.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of serine at position #₁ withtryptophan is designated as “Ser#₁Trp” or “S#₁W”. Multiple mutations areseparated by addition marks (“+”) or by commas (,), e.g.,“Ser#₁Trp+“Ser#₂Pro” or S#₁W, S#₂P, representing substitutions atpositions #₁ and #₂ of serine (S) with tryptophan (W) and proline (P),respectively. If more than one amino acid may be substituted in a givenposition these are listed in brackets, such as [X] or {X}. Thus if bothTrp and Lys according to the invention may be substituted instead of theamino acid occupying at position #₁ this is indicated as X#₁ {W, K} orX#₂ [W, K] where the X indicate that different proteases may be parente.g. such as a protease with SEQ ID NO 3 or a protease having at least70% identity hereto. Thus in some cases the variants are represented as#1 {W, K} or X#₂P indicating that the amino acids to be substituted varydepending on the parent.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofserine at position #₁ is designated as “Ser#₁*” or “S#₁*”. Multipledeletions are separated by addition marks (“+”) or commas, e.g.,“Ser#₁*+Ser#₂*” or “S#₁*, S#₂*”.

Insertions: The insertion of an additional amino acid residue such ase.g. a lysine after G#₁ may be indicated by: Gly#₁GlyLys or G#₁GK.Alternatively insertion of an additional amino acid residue such aslysine after G#₁ may be indicated by: *#₁aL. When more than one aminoacid residue is inserted, such as e.g. a Lys, and Ala after #₁ this maybe indicated as: Gly#₁GlyLysAla or G#₁GKA. In such cases, the insertedamino acid residue(s) may also be numbered by the addition of lower caseletters to the position number of the amino acid residue preceding theinserted amino acid residue(s), in this example: *#₁aK *#₁bA.

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”) or by commas (,), e.g.,“Ser#₁Trp+Ser#₂Pro” or “S#₁W, S#₂P” representing a substitution ofserine at positions #₁ and #₂ with tryptophan and proline, respectivelyas described above.

Different alterations: Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Ser#₁Trp, Lys” or S#₁W, K represents a substitution of serine atposition #₁ with tryptophan or lysine. Thus, “Ser#₁Trp, Lys+Ser#₂Asp”designates the following variants: “Ser#₁Trp+Ser#₂Pro”,“Ser#₁Lys+Ser#₂Pro” or S#₁W, K+S#₂D.

DETAILED DESCRIPTION OF THE INVENTION

Previously unanticipated, the inventors have found that proteasevariants containing an alteration at position 137 of SEQ ID NO: 3 and/orin the hydrophobic cluster around this position in SEQ ID NO: 3corresponding to the positions 121, 124 and 162 of SEQ ID NO: 3 haveimproved stability in detergent compared to a protease having theidentical amino acid sequence of said variant but not having thealteration(s) at one or more of said specified positions or compared toa protease with SEQ ID NO: 3. The positions corresponding to positions121, 124, 137 and 162 of SEQ ID NO: 3 form a hydrophobic cluster, whichis within approximately 4 Angstrom of position 137 as shown in Figure 1.Thus the present invention provides protease variants, comprising asubstitution at one or more (e.g., several) positions corresponding topositions 121, 124, 137 and 162, wherein the variant has proteaseactivity. New protease variants containing one or more substitution(s)in positions 121, 124, 137 and 162 (SEQ ID NO: 3 numbering), weregenerated and tested for stability in detergent as described in“Material and Methods” and the inventors demonstrate that one or moresubstitutions of one or more amino acid at a position corresponding topositions 121, 124, 137 and 162 of the mature polypeptide SEQ ID NO: 3significantly improved the detergent stability compared to a proteasehaving the identical amino acid sequence of said variant but not havinga substitution at one or more of said specified positions or compared toa protease with SEQ ID NO: 3. Surprisingly the variants according to theinvention may in addition to improved stability also have improved washperformance. Thus in a preferred embodiment the variants according tothe invention have improved detergent stability and/or improved washperformance compared to a protease having the identical amino acidsequence of said variant but not having a substitution at one or more ofsaid specified positions or compared to a protease with SEQ ID NO: 3. Ina preferred embodiment the protease variant comprises a substitution ofone or more amino acids at one more of the positions 121, 124, 137 and162 of SEQ ID NO: 3, wherein the variant has at least 70% identity tothe protease with SEQ ID NO: 3 (Bacillus sp. TY145). Thus one aspect ofthe invention relates to a protease variant, comprising a substitutionat one or more positions corresponding to positions 121, 124, 137 and162 of SEQ ID NO: 3, wherein the variant has an amino acid sequencewhich is at least 70% identical to SEQ ID NO 3; and recovering thevariant. The invention further relates to such variant comprisingsubstitution of one or more amino acids at one or more positionscorresponding to positions 121, 124, 137 and 162 of SEQ ID NO: 3,wherein the variant has at least 70%, such as at least 71%, such as atleast 72%, such as at least 73%, such as at least 74%, such as at least75%, at least 76% at least 77% at least 78% at least 79% at least 80%,at least 81% at least 82% at least 83% at least 84% at least 85%, atleast 86% at least 87% at least 88% at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95% identity, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to SEQ ID NO: 3. In one embodiment the variantis a polypeptide encoded by a polynucleotide having at least 70%identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or asequence encoding the mature polypeptide of SEQ ID NO: 2. In oneembodiment the variant according to the invention is a polypeptideencoded by a polynucleotide having at least 70% identity e.g., at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least76% at least 77% at least 78% at least 79% at least 80%, at least 81% atleast 82% at least 83% at least 84% at least 85%, at least 86% at least87% at least 88% at least 89%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95% identity, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the mature polynucleotide of SEQ ID NO: 1.

A particular embodiment, concerns a protease variant, comprising asubstitution at one or more positions corresponding to positions 121,124, 137 and 162 of SEQ ID NO: 3, wherein the variant is a variant of aparent protease which has at least 70%, such as at least 71%, such as atleast 72%, such as at least 73%, such as at least 74%, such as at least75%, e.g., such as at least 76% at least 77% at least 78% at least 79%at least 80%, at least 81% at least 82% at least 83% at least 84% atleast 85%, at least 86% at least 87% at least 88% at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 3. In one particular embodiment theprotease variant is a TY-145 (SEQ ID NO 3) variant comprising asubstitution of one or more amino acids in the hydrophobic clustercorresponding to positions 121, 124, 137 and 162 of SEQ ID NO: 3. Inanother embodiment, the invention relates to a variant comprising asubstitution at two, three, four or five positions corresponding topositions 121, 124, 137 and 162 of SEQ ID NO: 3. A preferred embodimentconcerns a protease variant, comprising substitution of one or moreamino acids in the hydrophobic cluster corresponding to positions 121,124, 137 and 162 of SEQ ID NO: 3, wherein the variant has at least 70%,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76% at least 77% at least 78% at least 79% at least 80%,at least 81% at least 82% at least 83% at least 84% at least 85%, atleast 86% at least 87% at least 88% at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94% at least 95% identity, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to SEQ ID NO: 3. A particularly preferredembodiment concerns a protease variant comprising one or more of thefollowing substitutions 121 {Ser, Cys, Val, Tyr}, 124 {Ala}, 137 {Glu,Cys, Ser, Ala, Met, Tyr, Gln, Gly} or 162 {Trp, Arg} of SEQ ID NO: 3. Aparticular embodiment concerns a protease variant comprising one or moreof the following substitutions 121 {Ser, Cys, Val, Tyr}, 124 {Ala}, 137{Glu, Cys, Ser, Ala, Met, Tyr, Gln, Gly} or 162 {Trp, Arg} of SEQ ID NO:3, wherein the variant has at least 70% identity to SEQ ID NO: 3 such asat least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%, butless than 100%, sequence identity to SEQ ID NO: 3. In one aspect, theprotease variant comprises a substitution at position 121, in apreferred aspect the variant comprises a substitution at position 121with S, C, V or T, in another preferred aspect, the variant comprises aS at position 121, in yet another preferred aspect, the variantcomprises the substitution I121 S, wherein the parent protease is themature polypeptide with SEQ ID NO: 3. In another preferred aspect, thevariant comprises an C at position 121, in yet another preferred aspect,the variant comprises the substitution I121C, wherein the parent is themature polypeptide with SEQ ID NO: 3, in another preferred aspect, thevariant comprises an V at position 121, in yet another preferred aspect,the variant comprises the substitution I121V, wherein the parent is themature polypeptide with SEQ ID NO: 3, in another preferred aspect, thevariant comprises an T at position 121, in yet another preferred aspect,the variant comprises the substitution I121T, wherein the parent is themature polypeptide with SEQ ID NO: 3.

In a further aspect, the variant comprises a substitution at position121 with S, C, V or T wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In another preferred aspect, the variantcomprises an S at position 121, in yet another preferred aspect, thevariant comprises the substitution I121S, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%. In another preferredaspect, the variant comprises an C at position 121, in yet anotherpreferred aspect, the variant comprises the substitution I121C, whereinthe variant has at least 70% sequence identity to SEQ ID NO: 3, such asat least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%. In anotherpreferred aspect, the variant comprises an V at position 121, in yetanother preferred aspect, the variant comprises the substitution I121V,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%. Inanother preferred aspect, the variant comprises an Tat position 121, inyet another preferred aspect, the variant comprises the substitutionI121T, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%.

In an even further aspect, the variant comprises a substitution atposition 121 with S, C, V or T wherein the variant has at least 70%sequence identity to SEQ ID NO: 3, such as at least 71%, at least 72%,at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in Example 2as described under “Material and Methods”. In another preferred aspect,the variant comprises a S at position 121, in yet another preferredaspect, the variant comprises the substitution I121 S, wherein thevariant has at least 70% sequence identity to SEQ ID NO: 3, such as atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, and havingan increased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in the Example 2 as described under “Material and Methods”. Inanother preferred aspect, the variant comprises an C at position 121, inyet another preferred aspect, the variant comprises the substitutionI121C, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, and having an increased stability relative to a protease with SEQID NO: 3 or a protease parent having the identical amino acid sequenceof said variant but not having the substitutions at one or more of saidpositions, when tested in the Example 2 as described under “Material andMethods”. In another preferred aspect, the variant comprises an V atposition 121, in yet another preferred aspect, the variant comprises thesubstitution I121V, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, and having an increased stability relative to aprotease with SEQ ID NO: 3 or a protease parent having the identicalamino acid sequence of said variant but not having the substitutions atone or more of said positions, when tested in the Example 2 as describedunder “Material and Methods”. In another preferred aspect, the variantcomprises an T at position 121, in yet another preferred aspect, thevariant comprises the substitution I121T, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In one aspect, the protease variant comprises a substitution at position124, in a preferred aspect the variant comprises a substitution atposition 124 with A in another preferred aspect, the variant comprisesthe substitution, wherein the parent is polypeptide with SEQ ID NO: 3.In another preferred aspect, the variant comprises an A at position 124,in yet another preferred aspect, the variant comprises the substitutionV124A, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%. In an even further aspect, the variant comprises a substitution atposition 124 with A, in yet another preferred aspect, the variantcomprises the substitution V124A, wherein the variant has at least 70%sequence identity to SEQ ID NO: 3, such as at least 71%, at least 72%,at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in Example 2as described under “Material and Methods”.

In one aspect, the protease variant comprises a substitution at position137, in a preferred aspect the variant comprises a substitution atposition 137 with E, C, S, A, M, T, Q or G. In another preferred aspect,the variant comprises a E at position 137, in yet another preferredaspect, the variant comprises the substitution I137E, wherein the parentis a polypeptide with SEQ ID NO: 3. In a further aspect, the variantcomprises a substitution at position 137 with E, in yet anotherpreferred aspect, the variant comprises the substitution I137E, whereinthe variant has at least 70% sequence identity to SEQ ID NO: 2, such asat least at least 71%, at least 72%, at least 73%, at least 74%, 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%. In afurther aspect, the variant comprises a substitution at position 137with E, in yet another preferred aspect, the variant comprises thesubstitution I137E, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, and having an increased stability relative to aprotease with SEQ ID NO: 3 or a protease parent having the identicalamino acid sequence of said variant but not having the substitutions atone or more of said positions, when tested in the Example 2 as describedunder “Material and Methods”

In another preferred aspect, the variant comprises a C at position 137,in yet another preferred aspect, the variant comprises the substitutionI137C, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with C, in yet another preferred aspect, the variant comprises thesubstitution I137C, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with C, in yet another preferred aspect,the variant comprises the substitution I137C, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises a S at position 137,in yet another preferred aspect, the variant comprises the substitutionI137S, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with S, in yet another preferred aspect, the variant comprises thesubstitution I137S, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with S, in yet another preferred aspect,the variant comprises the substitution I137S, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an A at position 137,in yet another preferred aspect, the variant comprises the substitutionI137A, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with A, in yet another preferred aspect, the variant comprises thesubstitution I137A, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with A, in yet another preferred aspect,the variant comprises the substitution I137A, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an M at position 137,in yet another preferred aspect, the variant comprises the substitutionI137M, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with M, in yet another preferred aspect, the variant comprises thesubstitution I137M, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with M, in yet another preferred aspect,the variant comprises the substitution I137M, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an T at position 137,in yet another preferred aspect, the variant comprises the substitutionI137T, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with T, in yet another preferred aspect, the variant comprises thesubstitution I137T, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with T, in yet another preferred aspect,the variant comprises the substitution I137T, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an Q at position 137,in yet another preferred aspect, the variant comprises the substitutionI137Q, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with Q, in yet another preferred aspect, the variant comprises thesubstitution I137Q, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with Q, in yet another preferred aspect,the variant comprises the substitution I137Q, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an G at position 137,in yet another preferred aspect, the variant comprises the substitutionI137G, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 137with G, in yet another preferred aspect, the variant comprises thesubstitution I137G, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a further aspect, the variant comprises asubstitution at position 137 with G, in yet another preferred aspect,the variant comprises the substitution I137G, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 2, such as at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, and havingan increased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in the Example 2 as described under “Material and Methods”

In one aspect, the protease variant comprises a substitution at position162, in a preferred aspect the variant comprises a substitution atposition 162 with W or R. In another preferred aspect, the variantcomprises a W at position 162, in yet another preferred aspect, thevariant comprises the substitution V162W, wherein the parent is apolypeptide with SEQ ID NO: 3. In a further aspect, the variantcomprises a substitution at position 162 with W, in yet anotherpreferred aspect, the variant comprises the substitution V162W, whereinthe variant has at least 70% sequence identity to SEQ ID NO: 3, such asat least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%. In anotherpreferred aspect, the variant comprises a substitution at position 162with W, in yet another preferred aspect, the variant comprises thesubstitution V162W, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, and having an increased stability relative to aprotease with SEQ ID NO: 3 or a protease parent having the identicalamino acid sequence of said variant but not having the substitutions atone or more of said positions, when tested in the Example 2 as describedunder “Material and Methods”.

In another preferred aspect, the variant comprises a R at position 162,in yet another preferred aspect, the variant comprises the substitutionV162R, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 162with R, in yet another preferred aspect, the variant comprises thesubstitution V162R, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In another preferred aspect, the variantcomprises a substitution at position 162 with R, in yet anotherpreferred aspect, the variant comprises the substitution V162R, whereinthe variant has at least 70% sequence identity to SEQ ID NO: 3, such asat least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, and havingan increased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in the Example 2 as described under “Material and Methods”.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121 and 124, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121 and 137, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121 and 162, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 124 and 137, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 124 and 162, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 137 and 162, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121, 124, and 137, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121, 124, and 162, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 124, 137, and 162, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 121, 124, 137, and 162, such as thosedescribed above.

In another aspect, the variant comprises one or more (several)substitutions selected from the group consisting of I121 {S, C, V, T},V124 {A}, I137 {E, C, S, A, M, T, Q, G} and V162 {W, R}.

In another aspect, the variant comprising the substitutions I121S+V124Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+V124Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+V124Aof the polypeptide with SEQ ID NO: 3

In another aspect, the variant comprising the substitutions I121T+V124Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Eof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Eof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Eof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Eof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Cof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Cof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Cof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Cof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Sof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Sof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Sof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Sof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Mof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Mof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Mof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Mof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Tof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Tof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Tof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Tof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Qof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Qof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Qof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Qof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+I137Gof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+I137Gof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+I137Gof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+I137Gof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121S+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121C+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121V+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I121T+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Eof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Cof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Sof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Mof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Tof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Qof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+I137Gof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions V124A+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137E+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137C+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137S+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137A+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137M+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137T+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137Q+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137G+V162Wof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137E+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137C+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137S+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137A+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137M+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137T+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137Q+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprising the substitutions I137G+V162Rof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137E of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137C of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137S of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137M of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137T of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137Q of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137G of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137E of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137C of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137S of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137M of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137T of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137Q of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137G of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137E of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137C of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137S of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137M of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137T of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137Q of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137G of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137E of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137C of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137S of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137M of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137T of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137Q of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137G of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137E+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137C+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137S+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137A+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137M+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137T+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137Q+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137G+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137E+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137C+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137S+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137A+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137M+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137T+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137Q+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsV124A+I137G+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137E+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137E+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137E+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137E+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137C+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137C+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137C+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137C+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137S+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137S+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137S+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137S+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137A+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137A+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137A+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137A+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137M+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137M+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137M+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137M+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137T+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137T+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137T+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137T+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137Q+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137Q+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137Q+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137Q+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137G+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137G+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137G+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137G+V162W of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137E+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137E+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137E+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137E+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137C+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137C+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137C+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137C+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137S+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137S+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137S+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137S+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137A+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137A+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137A+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137A+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137M+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137M+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137M+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137M+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137T+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137T+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137T+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137T+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137Q+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137Q+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137Q+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137Q+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121S+V124A+I137G+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121C+V124A+I137G+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121V+V124A+I137G+V162R of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsI121T+V124A+I137G+V162R of the polypeptide with SEQ ID NO: 3.

The variant may further comprise a substitution at one or more (several)other positions. For example, the variants may comprise a substitutionat one or more positions corresponding to positions selected from thegroup consisting of 39, 40, 70, 74, 81, 102, 132, 144, 155, 159, 171,173, 174, 175, 176, 177, 179, 180, 241, 247, 256, 274, 286 and 297. Inone embodiment the variant of the invention comprises one or more of thefollowing alterations: Y39D; T40{D,P}; Q70N; T74M; L81{F,H,V}; A102T;G132 {I,E}; S144{Q,R}; D155N; G159S; S171 {W, K, E}; S173{P,V};G174{S,T}; S175 {A, V, P}; N176G; T177S; G179 {C, V, Q, S, T, E, H, K,M, N}; F180Y; T241P; I247M; H256F; S274I; V286Q or T297P.

In another aspect, a variant according to the invention comprises asubstitution at one or more (e.g., several) positions corresponding topositions 121, 124, 137 and 162. In another aspect, a variant accordingto the invention comprises a substitution at two positions correspondingto any of positions 121, 124, 137 and 162. In another aspect, a variantaccording to the invention comprises a substitution at three positionscorresponding to any of positions 121, 124, 137 and 162. In anotheraspect, a variant according to the invention comprises a substitution ateach position corresponding to positions 121, 124, 137 and 162.

The variants may further comprise one or more additional alterations atone or more (e.g., several) other positions. The amino acid changes maybe of a minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of 1-30 amino acids; smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Asn/Gln, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Glu/Gln, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

For example, the variants may comprise an substitution at a positioncorresponding to any of the positions 121, 124, 137 and 162 and furthercomprises one or more alteration at any of the positions selected fromthe group consisting of positions 39, 40, 70, 74, 81, 102, 132, 144,155, 159, 171, 173, 174, 175, 176, 177, 179, 180, 241, 247, 256, 274,286 and 297. In a preferred embodiment the alteration at any of thepositions selected from the group consisting of 39, 40, 70, 74, 81, 102,132, 144, 155, 159, 171, 173, 174, 175, 176, 177, 179, 180, 241, 247,256, 274, 286 and 297 is a substitution. In a particular preferredembodiment the variants according to the invention comprises any of thefollowing substitutions I121 {S, C, V, T}, V124A, I137 {E, C, S, A, M,T, Q, G} and V162{W, R} of SEQ ID NO: 3, wherein the variant furthercomprises one or more substitution selected from the group consistingof: L34I, Y39D; T40{D,P,L}; Q70N; T74M; L81{F,H,V}; A102T; R130A, G132{I,E}; S133T S144{Q,R}; D155N; G159S; V162R, S171 {W, K, E, N};5173{P,V}; G174{S,T}; S175 {A, V, P}; N176G; T177S; G179 {C, V, Q, S, T,E, H, K, M, N, A, Y}; F180Y; T241P; I247M; H256F, S274I; V286Q or T297P

In one embodiment of the invention, the variants according to theinvention comprise or consist of any of the following variants:

I137M S173P I121V S175P I137E S175P I121V S175A I137E S175A I137E S144QI137E S144R I137M S144R I121T S175P I137M S144Q V124A S133T V124A R130AI137E S173Y G174S S175A F180Y V162R S173P G174T S175V T177S F180Y I121VS173P G174T S175V T177S F180Y V162R S173P G174K S175P N176G T177S F180YI121V S173P G174K S175P N176G T177S F180Y I137E S173P G174K S175P N176GT177S F180Y I121V I137E S173Y G174S S175A F180Y L81V I137E S173Y G174SS175A F180Y I137E S173Y G174S S175A F180Y T241P Q70N I137E S173Y G174SS175A F180Y I137E S173Y G174S S175A F180Y S274I I137E S173Y G174S S175AF180Y T297P I137E S173P G174T S175V T177S F180Y T241P I137E S173P G174TS175V T177S F180Y V286Q I137E S171N S173P G174T S175V T177S F180Y I137ES173P G174T S175A T177S F180Y I121V I137E S173P G174K S175P N176G T177SF180Y Q70N I137E S173P G174K S175P N176G T177S F180Y I137E S173P G174KS175P N176G T177S F180Y S274I I137E S173P G174K S175P N176G T177S F180YV286Q I137E S173P G174K S175P N176G T177S F180Y T297P I137E S171N S173PG174K S175P N176GT177S F180Y I137E S173P G174K S175A N176G T177S F180YV162R S173Y G174S S175A F180Y I121V S173Y G174S S175A F180Y I121V S144QV162R S173P G174T S175V T177S F180Y L81V I137E S173Y G174S S175A F180YI121V I137E S173P G174T S175V T177S F180Y I137E S173P G174T S175V T177SF180Y T297P I137E S171N S173P G174T S175V T177S F180Y V162R S173Y G174SS175A F180Y I137E S144Q S173Y G174S S175A F180Y T40L I137E S173Y G174SS175A F180Y I137E S173Y S175A F180Y I137E S173P S175P N176G T177S F180YI137E S173Y G174S S175A F180Y V286Q I137E S173P G174T S175V T177S F180YS274I T74M I137E S173P L34I I137E S173P Y39D I137E S173P T40P I137ES173P I137E S173P I247M I137E S173P H256F I137E S144Q S173P G174T S175VT177S F180Y I137E S144Q S173P G174K S175P N176G T177S F180Y I137E S173YG174S S175P F180Y V162R S171N S173P G174K S175P N176G T177S F180Y I137ES171N S173P G174K S175P N176G T177S F180Y T241P I137E S173P S175P F180YV162R S173P S175P F180Y I137E S173P S175V T177S F180Y

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide. ForTY-145 (SEQ ID NO: 3) the catalytic triad comprising the amino acidsD35, H72 and S251 is essential for protease activity of the enzyme. Thevariants may consist of 200 to 900 amino acids, e.g., 210 to 800, 220 to700, 230 to 600, 240 to 500, 250 to 400, 255 to 300, 260 to 290, 265 to285, 270 to 280 or 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or280 amino acids.

In an embodiment, the variant has improved catalytic activity comparedto the parent enzyme.

In an embodiment, the variant has improved stability compared to theparent enzyme or compared to a protease having the identical amino acidsequence of said variant but not having a substitution at one or more ofsaid specified positions or compared to a protease with SEQ ID NO: 3,wherein stability is measured in Example 2 as described in “Material andMethods” herein.

In an embodiment, a variant according to the invention has improvedstability compared to the parent enzyme or compared to a protease havingthe identical amino acid sequence of said variant but not having asubstitution at one or more of said specified positions or compared to aprotease with SEQ ID NO: 3.

Parent Proteases

Protease Variant

The term “variant” and the term “protease variant” are defined above.

Homologous Subtilase Sequences

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the artto identify suitable homologous subtilases, which can be modifiedaccording to the invention.

Substantially homologous parent subtilase variants may have one or more(several) amino acid substitutions, deletions and/or insertions, in thepresent context the term “one or more” is used interchangeably with theterm “several”. These changes are preferably of a minor nature, that isconservative amino acid substitutions as described above and othersubstitutions that do not significantly affect the three-dimensionalfolding or activity of the protein or polypeptide; small deletions,typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification (an affinity tag), such asa poly-histidine tract, or protein A (Nilsson et al., 1985, EMBO J. 4:1075; Nilsson et al., 1991, Methods Enzymol. 198: 3. See, also, ingeneral, Ford et al., 1991, Protein Expression and Purification 2:95-107.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

The parent subtilase may comprise or consist of the amino acid sequenceof SEQ ID NO: 3 or an allelic variant thereof; or a fragment thereofhaving protease activity. In one aspect, the parent subtilase comprisesor consists of the amino acid sequence of SEQ ID NO: 3.

The parent subtilase may be (a) a polypeptide having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 3; (b) apolypeptide encoded by a polynucleotide that hybridizes under medium orhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii); or (c) a polypeptide encoded by a polynucleotide having atleast 70% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the polypeptide withSEQ ID NO: 3 of at least 70%, such as at least 71%, at least 72%, atleast 73%, at least 74% at least 75%, at least 76% at least 77% at least78% at least 79% at least 80%, at least 81% at least 82% at least 83% atleast 84% at least 85%, at least 86% at least 87% at least 88% at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94% at least 95% identity, at least 96%, at least 97%, at least 98%, orat least 99%, or 100%, which have protease activity.

In one aspect, the amino acid sequence of the parent differs by no morethan 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the maturepolypeptide with SEQ ID NO: 3.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3. In another aspect, the parent comprises orconsists of amino acids 1 to 311 of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, or high stringency conditions,or very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii), (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 3 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) a sequence encoding the mature polypeptide of SEQ ID NO:2; (iv) the full-length complement thereof; or (v) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleotide acid probeis a 80 to 1140 nucleotides long fragment of SEQ ID NO: 1, e.g. 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long.In another aspect, the nucleic acid probe is a polynucleotide thatencodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof;or a fragment thereof. In another aspect, the nucleic acid probe is SEQID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2 atleast 70%, e.g., at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 76%, at least 77% at least 78% at least 79% atleast 80%, at least 81% at least 82% at least 83% at least 84% at least85%, at least 86% at least 87% at least 88% at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from organisms of any genus. For purposes ofthe present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the parent encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, theparent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease

In one aspect, the parent is a Bacillus sp. protease, e.g., the proteasewith SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO 2.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving protease activity, comprising: (a) introducing into a parentsubtilase a substitution at one or more (e.g., several) positionscorresponding to positions 121, 124, 137 or 162 of SEQ ID NO: 3, whereinthe variant has protease activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Thus, the invention also relates to a method for obtaining a proteasevariant, comprising introducing into a parent protease a substitution atone or more positions corresponding to positions 121, 124, 137 or 162 ofSEQ ID NO: 3; and recovering the variant.

Another embodiment concerns a method for obtaining a protease variant,comprising substitution of one or more amino acids in the hydrophobiccluster corresponding to positions 121, 124, 137 or 162 of SEQ ID NO: 3,especially a method as described above, wherein the parent protease isselected from the group consisting of: a polypeptide having at least 70%sequence identity to SEQ ID NO: 3;

a. a polypeptide encoded by a polynucleotide that hybridizes undermedium or high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii);

b. a polypeptide encoded by a polynucleotide having at least 70%identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or asequence encoding the mature polypeptide of SEQ ID NO: 2; and

c. a fragment of the mature polypeptide of SEQ ID NO: 2, which hasprotease activity. Thus, a particular aspect concerns a method forobtaining a protease variant, comprising introducing into a parentprotease a substitution of one or more amino acids in the hydrophobiccluster corresponding to positions 121, 124, 137 or 162 of SEQ ID NO: 3,wherein the substitution(s) is/are performed in SEQ ID NO: 3, andwherein the substitutions are selected from the group consisting ofsubstitutions I121 {S, C, V, T}, V124A, I137 {E, C, S, A, M, T, Q, G}and V162{W, R}.

One aspect of the invention relates to methods of producing the variantsaccording to the invention, wherein the method comprises substitution ofat least one amino acid in the hydrophobic cluster corresponding topositions 121, 124, 137 or 162 of SEQ ID NO: 3, wherein

(a) the variant has a sequence identity to SEQ ID NO: 3 of at least 70%and less than 100% and

(b) the variant has protease activity.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 121 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 124, 137 and 162 of SEQ ID NO: 3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 124 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 121, 137 and 162 of SEQ ID NO: 3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 137 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 121, 124 and 162 of SEQ ID NO: 3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 162 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 121, 124 and 137 of SEQ ID NO: 3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 121 ofSEQ ID NO: 3 with an amino acid selected from {S, C, V, T}.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 124 ofSEQ ID NO: 3 with A.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 137 ofSEQ ID NO: 3 with an amino acid selected from {E, C, S, A, M, T, Q, G}.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 162 ofSEQ ID NO: 3 with an amino acid selected from {W, R}.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

In one certain aspect, the variants according to the invention hasimproved stability in detergents compared to the parent enzyme orcompared to a protease having the identical amino acid sequence of saidvariant but not having the substitutions at one or more of saidspecified positions or compared to a protease with SEQ ID NO 3, whereinstability is measured in Example 2 as described in “Material andMethods” herein.

Thus, in a preferred embodiment the composition is a detergentcomposition, and one aspect of the invention relates to the use of adetergent composition comprising a variant according to the invention ina cleaning process such as laundry or hard surface cleaning.

The choice of additional components is within the skill of the artisanand includes conventional ingredients, including the exemplarynon-limiting components set forth below. The choice of components mayinclude, for fabric care, the consideration of the type of fabric to becleaned, the type and/or degree of soiling, the temperature at whichcleaning is to take place, and the formulation of the detergent product.Although components mentioned below are categorized by general headeraccording to a particular functionality, this is not to be construed asa limitation, as a component may comprise additional functionalities aswill be appreciated by the skilled artisan.

Enzyme of the Present Invention

In one embodiment of the present invention, the variants of the presentinvention may be added to a detergent composition in an amountcorresponding to 0.001-100 mg of protein, such as 0.01-100 mg ofprotein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mgof protein, even more preferably 0.05-10 mg of protein, most preferably0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein perliter of wash liquor.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example,WO92/19709 and WO92/19708 or the variants according to the invention maybe stabilized using peptide aldehydes or ketones such as described inWO2005/105826 and WO2009/118375.

A variant of the present invention may also be incorporated in thedetergent formulations disclosed in WO97/07202, which is herebyincorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein, the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylehanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, and combinationsthereof, Alkyl quaternary ammonium compounds, Alkoxylated quaternaryammonium (AQA).

When included therein, the detergent will usually contain from about0.2% to about 40% by weight of a non-ionic surfactant, for example fromabout 0.5% to about 30%, in particular from about 1% to about 20%, fromabout 3% to about 10%, such as from about 3% to about 5%, or from about8% to about 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein, the detergent will usually contain from about 1%to about 40% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, and sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluenesulfonates (STS), sodium xylene sulfonates (SXS), sodium cumenesulfonates (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 50% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry detergents may be utilized. Non-limiting examples of buildersinclude zeolites, diphosphates (pyrophosphates), triphosphates such assodium triphosphate (STP or STPP), carbonates such as sodium carbonate,soluble silicates such as sodium metasilicate, layered silicates (e.g.,SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA),iminodiethanol (DEA) and 2,2′,2″-nitrilotriethanol (TEA), andcarboxymethylinulin (CMI), and combinations thereof.

The detergent composition may also contain 0-65% by weight, such asabout 5% to about 40%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO 09/102854, U.S. Pat. No. 5,977,053.

Bleaching Systems

The detergent may contain 0-10% by weight, such as about 1% to about 5%,of a bleaching system. Any bleaching system known in the art for use inlaundry detergents may be utilized. Suitable bleaching system componentsinclude bleaching catalysts, photobleaches, bleach activators, sourcesof hydrogen peroxide such as sodium percarbonate and sodium perborates,preformed peracids and mixtures thereof. Suitable preformed peracidsinclude, but are not limited to, peroxycarboxylic acids and salts,percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone (R), and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. By bleach activator is meant herein a compound whichreacts with peroxygen bleach like hydrogen peroxide to form a peracid.The peracid thus formed constitutes the activated bleach. Suitablebleach activators to be used herein include those belonging to the classof esters amides, imides or anhydrides. Suitable examples are tetracetylathylene diamine (TAED), sodium 3,5,5 trimethyl hexanoyloxybenzenesulphonat, diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate(LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS),tetraacetylethylenediamine (TAED) and 4-(nonanoyloxy)benzenesulfonate(NOBS), and/or those disclosed in WO98/17767. A particular family ofbleach activators of interest was disclosed in EP624154 and particularlypreferred in that family is acetyl triethyl citrate (ATC). ATC or ashort chain triglyceride like Triacin has the advantage that it isenvironmental friendly as it eventually degrades into citric acid andalcohol. Furthermore acethyl triethyl citrate and triacetin has a goodhydrolytical stability in the product upon storage and it is anefficient bleach activator. Finally ATC provides a good buildingcapacity to the laundry additive. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthaloylamino)percapronic acid (PAP). The bleaching system may alsoinclude a bleach catalyst. In some embodiments the bleach component maybe an organic catalyst selected from the group consisting of organiccatalysts having the following formulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO2007/087258, WO2007/087244, WO2007/087259, WO2007/087242. Suitablephotobleaches may for example be sulfonated zinc phthalocyaninePolymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of polyethyleneterephthalate and polyoxyethene terephthalate (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridin-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present invention may also includefabric hueing agents such as dyes or pigments which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions thusaltering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and

Basic Red, or mixtures thereof, for example as described inWO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (herebyincorporated by reference). The detergent composition preferablycomprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt % to0.2 wt % fabric hueing agent, this may be especially preferred when thecomposition is in the form of a unit dose pouch. Suitable hueing agentsare also disclosed in, e.g., WO 2007/087257, WO2007/087243.

(Additional) Enzymes

In one embodiment, the variants according to the invention are combinedwith one or more enzymes, such as at least two enzymes, more preferredat least three, four or five enzymes. Preferably, the enzymes havedifferent substrate specificity, e.g., proteolytic activity, amylolyticactivity, lipolytic activity, hemicellulytic activity or pectolyticactivity.

The detergent additive as well as the detergent composition may compriseone or more additional enzymes such as carbohydrate-active enzymes likecarbohydrase, pectinase, mannanase, amylase, cellulase, arabinase,galactanase, xylanase, or protease, lipase, a, cutinase, oxidase, e.g.,a laccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Mannanases

Suitable mannanases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. The mannanasemay be an alkaline mannanase of Family 5 or 26. It may be a wild-typefrom Bacillus or Humicola, particularly B. agaradhaerens, B.licheniformis, B. halodurans, B. clausii, or H. insolens. Suitablemannanases are described in WO 1999/064619. A commercially availablemannanase is Mannaway (Novozymes A/S).

Cellulase:

Suitable cellulases include complete cellulases or mono-componentendoglucanases of bacterial or fungal origin. Chemically or geneticallymodified mutants are included. The cellulase may for example be amono-component or a mixture of mono-component endo-1,4-beta-glucanaseoften just termed endoglucanases. Suitable cellulases include a fungalcellulase from Humicola insolens (U.S. Pat. No. 4,435,307) or fromTrichoderma, e.g. T. reesei or T. viride. Examples of cellulases aredescribed in EP 0 495 257. Other suitable cellulases are from Thielaviae.g. Thielavia terrestris as described in WO 96/29397 or Fusariumoxysporum as described in WO 91/17244 or from Bacillus as described in,WO 02/099091 and JP 2000210081. Other examples are cellulase variantssuch as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No.5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO95/24471, WO 98/12307 Commercially available cellulases includeCarezyme®, Celluzyme®, Celluclean®, Celluclast® and Endolase®;Renozyme®; Whitezyme® (Novozymes NS) Puradax®, Puradax HA, and PuradaxEG (available from Genencor).

Cellulases:

Suitable cellulases include complete cellulases or mono-componentendoglucanases of bacterial or fungal origin. Chemically or geneticallymodified mutants are included. The cellulase may for example be amono-component or a mixture of mono-component endo-1,4-beta-glucanaseoften just termed endoglucanases. Suitable cellulases include a fungalcellulase from Humicola insolens (U.S. Pat. No. 4,435,307) or fromTrichoderma, e.g. T. reesei or T. viride. Examples of cellulases aredescribed in EP 0 495 257. Other suitable cellulases are from Thielaviae.g. Thielavia terrestris as described in WO 96/29397 or Fusariumoxysporum as described in WO 91/17244 or from Bacillus as described in,WO 02/099091 and JP 2000210081. Other examples are cellulase variantssuch as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No.5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO95/24471, WO 98/12307 Commercially available cellulases includeCarezyme®, Celluzyme®, Celluclean®, Celluclast® and Endolase®;Renozyme®; Whitezyme® (Novozymes NS) Puradax®, Puradax HA, and PuradaxEG (available from Genencor).

Proteases:

Suitable proteases include those of bacterial, fungal, plant, viral oranimal origin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellulomonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens. Examples of useful proteases are thevariants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116,WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979,WO07/006305, WO11/036263, WO11/036264, especially the variants withsubstitutions in one or more of the following positions: 3, 4, 9, 15,27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205,206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using theBPN′ numbering. More preferred the subtilase variants may comprise themutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E,A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R,H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E,V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K,T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Preferenz™, Purafect MA®, Purafect Ox®), Purafect OxP®, Puramax®,Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Eraser®,Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.),BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variantshereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases:

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases:

Suitable amylases which can be used together with the protease variantsof the invention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/023873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions:K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459,D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are thosehaving the substitution in one of more of the following positions:K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T,G476K and G477K and/or deletion in position R178 and/or S179 or of T180and/or G181. Most preferred amylase variants of SEQ ID NO: 1 are thosehaving the substitutions:

E187P+I203Y+G476K

E187P+I203Y+R458N+T459S+D460T+G476K,

wherein the variants optionally further comprises a substitution atposition 241 and/or a deletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions: N21,D97, V128I K177, R179, S180, I181, G182, M200, L204, E242, G477 andG478. More preferred variants of SEQ ID NO: 1 are those having thesubstitution in one of more of the following positions: N21D, D97N,V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion inposition R179 and/or S180 or of I181 and/or G182. Most preferred amylasevariants of SEQ ID NO: 1 are those having the substitutions:

N21D+D97N+V128I,

wherein the variants optionally further comprises a substitution atposition 200 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes NS), and Rapidase™ Purastar/Effectenz™, Powerase, PreferenzS1000, Preferenz S100 and Preferenz S110 (from Genencor InternationalInc./DuPont).

Peroxidases/Oxidases:

A peroxidase according to the invention is a peroxidase enzyme comprisedby the enzyme classification EC 1.11.1.7, as set out by the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (IUBMB), or any fragment derived therefrom, exhibitingperoxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinopsis, e.g., fromC. cinerea (EP 179,486), and variants thereof as those described in WO93/24618, WO 95/10602, and WO 98/15257.

A peroxidase according to the invention also includes a haloperoxidaseenzyme, such as chloroperoxidase, bromoperoxidase and compoundsexhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidasesare classified according to their specificity for halide ions.Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochloritefrom chloride ions.

In an embodiment, the haloperoxidase of the invention is achloroperoxidase. Preferably, the haloperoxidase is a vanadiumhaloperoxidase, i.e., a vanadate-containing haloperoxidase. In apreferred method of the present invention the vanadate-containinghaloperoxidase is combined with a source of chloride ion.

Haloperoxidases have been isolated from many different fungi, inparticular from the fungus group dematiaceous hyphomycetes, such asCaldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C.verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such asPseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.aureofaciens.

In an preferred embodiment, the haloperoxidase is derivable fromCurvularia sp., in particular Curvularia verruculosa or Curvulariainaequalis, such as C. inaequalis CBS 102.42 as described in WO95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 asdescribed in WO 97/04102; or from Drechslera hartlebii as described inWO 01/79459, Dendryphiella salina as described in WO 01/79458,Phaeotrichoconis crotalarie as described in WO 01/79461, orGeniculosporium sp. as described in WO 01/79460.

An oxidase according to the invention include, in particular, anylaccase enzyme comprised by the enzyme classification EC 1.10.3.2, orany fragment derived therefrom exhibiting laccase activity, or acompound exhibiting a similar activity, such as a catechol oxidase (EC1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), or a bilirubinoxidase (EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymesmay be derived from plants, bacteria or fungi (including filamentousfungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred; inparticular a laccase derived from Coprinopsis cinerea, as disclosed inWO 97/08325; or from Myceliophthora thermophila, as disclosed in WO95/33836.

Other Enzymes:

A protease variant according to the invention may also be combined withadditional enzymes such as pectate lyases e.g. Pectawash™,chlorophyllases etc. The protease variant of the invention may be mixedwith any additional enzyme.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants—The detergent compositions of the present invention can alsocontain dispersants. In particular powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents—The detergent compositions of the presentinvention may also include one or more dye transfer inhibiting agents.Suitable polymeric dye transfer inhibiting agents include, but are notlimited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a subject composition, the dye transfer inhibiting agents maybe present at levels from about 0.0001% to about 10%, from about 0.01%to about 5% or even from about 0.1% to about 3% by weight of thecomposition.

Fluorescent whitening agent—The detergent compositions of the presentinvention will preferably also contain additional components that maytint articles being cleaned, such as fluorescent whitening agent oroptical brighteners. Where present the brightener is preferably at alevel of about 0.01% to about 0.5%. Any fluorescent whitening agentsuitable for use in a laundry detergent composition may be used in thecomposition of the present invention. The most commonly used fluorescentwhitening agents are those belonging to the classes ofdiaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivativesand bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulphonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diaryl pyrazolines andthe 7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

Soil release polymers—The detergent compositions of the presentinvention may also include one or more soil release polymers which aidthe removal of soils from fabrics such as cotton and polyester basedfabrics, in particular the removal of hydrophobic soils from polyesterbased fabrics. The soil release polymers may for example be nonionic oranionic terephthalte based polymers, polyvinyl caprolactam and relatedcopolymers, vinyl graft copolymers, polyester polyamides see for exampleChapter 7 in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc. Another type of soil release polymers areamphiphilic alkoxylated grease cleaning polymers comprising a corestructure and a plurality of alkoxylate groups attached to that corestructure. The core structure may comprise a polyalkylenimine structureor a polyalkanolamine structure as described in detail in WO 2009/087523(hereby incorporated by reference). Furthermore random graft co-polymersare suitable soil release polymers Suitable graft co-polymers aredescribed in more detail in WO 2007/138054, WO 2006/108856 and WO2006/113314 (hereby incorporated by reference). Other soil releasepolymers are substituted polysaccharide structures especiallysubstituted cellulosic structures such as modified cellulosederiviatives such as those described in EP 1867808 or WO 2003/040279(both are hereby incorporated by reference). Suitable cellulosicpolymers include cellulose, cellulose ethers, cellulose esters,cellulose amides and mixtures thereof. Suitable cellulosic polymersinclude anionically modified cellulose, nonionically modified cellulose,cationically modified cellulose, zwitterionically modified cellulose,and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethylcellulose, hydroxyl propyl methyl cellulose, ester carboxy methylcellulose, and mixtures thereof.

Anti-redeposition agents—The detergent compositions of the presentinvention may also include one or more anti-redeposition agents such ascarboxymethylcellulose (CMC), polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol(PEG), homopolymers of acrylic acid, copolymers of acrylic acid andmaleic acid, and ethoxylated polyethyleneimines. The cellulose basedpolymers described under soil release polymers above may also functionas anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,structurants for liquid detergents and/or structure elasticizing agents.

Formulation of Detergent Products

The detergent composition of the invention may be in any convenientform, e.g., a bar, a homogenous tablet, a tablet having two or morelayers, a regular or compact powder, a granule, a paste, a gel, or aregular, compact or concentrated liquid.

Detergent formulation forms: Layers (same or different phases), Pouches,versus forms for Machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxyprpyl methyl cellulose (HPMC). Preferably the level of polymer inthe film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blend compositions comprising hydrolytically degradableand water soluble polymer blends such as polyactide and polyvinylalcohol (known under the Trade reference M8630 as sold by Chris CraftIn. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethyleneglycerol, Propylene glycol, sorbitol and mixtures thereof. The pouchescan comprise a solid laundry cleaning composition or part componentsand/or a liquid cleaning composition or part components separated by thewater soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. Ref:(US2009/0011970 A1)

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

Definition/Characteristics of the Forms:

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO09/092699,EP1705241, EP1382668, WO07/001262, US6472364, WO04/074419 orWO09/102854. Other useful detergent formulations are described inWO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342,WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296,WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125,WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375,WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545,WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391,WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770,WO09/021813, WO09/030632, and WO09/015951.

WO2011025615, WO2011016958, WO2011005803, WO2011005623, WO2011005730,WO2011005844, WO2011005904, WO2011005630, WO2011005830, WO2011005912,WO2011005905, WO2011005910, WO2011005813, WO2010135238, WO2010120863,WO2010108002, WO2010111365, WO2010108000, WO2010107635, WO2010090915,WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979,WO2010030540, WO2010030541, WO2010030539, WO2010024467, WO2010024469,WO2010024470, WO2010025161, WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051, WO2010102861, WO2010099997,WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957,WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO2011023716, WO2010142539, WO2010118959, WO2010115813, WO2010105942,WO2010105961, WO2010105962, WO2010094356, WO2010084203, WO2010078979,WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486,WO2010066631, WO2010066632, WO2010063689, WO2010060821, WO2010049187,WO2010031607, WO2010000636,

Methods and Uses

The present invention is also directed to methods for using thecompositions thereof in laundry of textile and fabrics, such as household laundry washing and industrial laundry washing.

The invention is also directed to methods for using the compositionsthereof in hard surface cleaning such as automated Dish Washing (ADW),car wash and cleaning of Industrial surfaces.

The protease variants of the present invention may be added to and thusbecome a component of a detergent composition. Thus one aspect of theinvention relates to the use of a detergent composition comprising aprotease variant wherein said variant comprises a substitution of one ormore amino acids in the hydrophobic cluster corresponding to positions121, 124, 137 and 162 of SEQ ID NO: 3, wherein the variant has at least70% identity to SEQ ID NO: 3 in a cleaning process such as laundryand/or hard surface cleaning e.g. dish wash. Another aspect relates tothe use of a detergent composition comprising a variant wherein saidvariant comprises one or more of the following substitutions I121 {S, C,V, T}, V124A, I137 {E, C, S, A, M, T, G} and V162{W, R} of SEQ ID NO: 3,wherein the variant has a sequence identity to SEQ ID NO: 3 of at least70% and less than 100% and the variant has protease activity.

One embodiment of the invention relates to the use of a proteasevariant, comprising one or more of the following substitutions I121 {S,C, V, T}, V124A, I137 {E, C, S, A, M, T, Q, G} and V162{W, R} of SEQ IDNO: 3, wherein the variant has at least 70% such as at least 71%, suchas at least 72%, such as at least 73%, such as at least 74%, such as atleast 75%, at least 76% at least 77% at least 78% at least 79% at least80%, at least 81% at least 82% at least 83% at least 84% at least 85%,at least 86% at least 87% at least 88% at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%, butless than 100%, sequence identity to SEQ ID NO: 3 in a cleaning processsuch as laundry and/or hard surface cleaning and wherein the variant hasincreased detergent stability relative to the parent or relative to aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions whentested in the Example 2, as described under “Material and Methods”.

A detergent composition of the present invention may be formulated, forexample, as a hand or machine laundry detergent composition including alaundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the present invention as describedherein.

The cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. Furthermore, the invention relates to aprocess for laundering of fabrics and/or garments where the processcomprises treating fabrics with a washing solution containing adetergent composition, and at least one protease variant of theinvention. The cleaning process or a textile care process can forexample be carried out in a machine washing process or in a manualwashing process. The washing solution can for example be an aqueouswashing solution containing a detergent composition.

The fabrics and/or garments subjected to a washing, cleaning or textilecare process of the present invention may be conventional washablelaundry, for example household laundry. Preferably, the major part ofthe laundry is garments and fabrics, including knits, woven, denims,non-woven, felts, yarns, and towelling. The fabrics may be cellulosebased such as natural cellulosics, including cotton, flax, linen, jute,ramie, sisal or coir or manmade cellulosics (e.g., originating from woodpulp) including viscose/rayon, ramie, cellulose acetate fibers(tricell), lyocell or blends thereof. The fabrics may also benon-cellulose based such as natural polyamides including wool, camel,cashmere, mohair, rabbit and silk or synthetic polymer such as nylon,aramid, polyester, acrylic, polypropylen and spandex/elastane, or blendsthereof as well as blend of cellulose based and non-cellulose basedfibers. Examples of blends are blends of cotton and/or rayon/viscosewith one or more companion material such as wool, synthetic fibers(e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinylalcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyureafibers, aramid fibers), and cellulose-containing fibers (e.g.,rayon/viscose, ramie, flax, linen, jute, cellulose acetate fibers,lyocell).

The last few years there has been an increasing interest in replacingcomponents in detergents, which is derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change new enzyme activities or new enzymes havingalternative and/or improved properties compared to the common useddetergent enzymes such as proteases, lipases and amylases is needed toachieve a similar or improved wash performance when compared to thetraditional detergent compositions.

The invention further concerns the use of protease variants of theinvention in a proteinaceous stain removing processes. The proteinaceousstains may be stains such as food stains, e.g., baby food, sebum, cocoa,egg, blood, milk, ink, grass, or a combination hereof.

Typical detergent compositions include various components in addition tothe enzymes, these components have different effects, some componentslike the surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away, other components like bleach systems remove discolor oftenby oxidation and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Yet other components likebuilder and chelator softens, e.g., the wash water by removing the metalions form the liquid.

In a particular embodiment, the invention concerns the use of acomposition comprising a protease variant of the invention, wherein saidenzyme composition further comprises at least one or more of thefollowing: a surfactant, a builder, a chelator or chelating agent,bleach system or bleach component in laundry or dish wash.

In a preferred embodiment of the invention, the amount of a surfactant,a builder, a chelator or chelating agent, bleach system and/or bleachcomponent are reduced compared to amount of surfactant, builder,chelator or chelating agent, bleach system and/or bleach component usedwithout the added protease variant of the invention. Preferably the atleast one component which is a surfactant, a builder, a chelator orchelating agent, bleach system and/or bleach component is present in anamount that is 1% less, such as 2% less, such as 3% less, such as 4%less, such as 5% less, such as 6% less, such as 7% less, such as 8%less, such as 9% less, such as 10% less, such as 15% less, such as 20%less, such as 25% less, such as 30% less, such as 35% less, such as 40%less, such as 45% less, such as 50% less than the amount of thecomponent in the system without the addition of protease variants of theinvention, such as a conventional amount of such component. In oneaspect, a protease variant of the invention is used in detergentcompositions wherein said composition is free of at least one componentwhich is a surfactant, a builder, a chelator or chelating agent, bleachsystem or bleach component and/or polymer.

Washing Method

The detergent compositions of the present invention are ideally suitedfor use in laundry applications. Accordingly, the present inventionincludes a method for laundering a fabric. The method comprises thesteps of contacting a fabric to be laundered with a cleaning laundrysolution comprising the detergent composition according to theinvention. The fabric may comprise any fabric capable of being launderedin normal consumer use conditions. The solution preferably has a pH fromabout 5.5 to about 11.5. The compositions may be employed atconcentrations from about 100 ppm, preferably 500 ppm to about 15,000ppm in solution. The water temperatures typically range from about 5° C.to about 95° C., including about 10° C., about 15° C., about 20° C.,about 25° C., about 30° C., about 35° C., about 40° C., about 45° C.,about 50° C., about 55° C., about 60° C., about 65° C., about 70° C.,about 75° C., about 80° C., about 85° C. and about 90° C. The water tofabric ratio is typically from about 1:1 to about 30:1.

In particular embodiments, the washing method is conducted at a pH fromabout 5.0 to about 11.5, or from about 6 to about 10.5, about 5 to about11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 toabout 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 toabout 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about11, about 7 to about 10, about 7 to about 9, or about 7 to about 8,about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 toabout 11, about 9 to about 10, about 10 to about 11, preferably about5.5 to about 11.5.

In particular embodiments, the washing method is conducted at a degreeof hardness of from about 0° dH to about 30° dH, such as about 1° dH,about 2° dH, about 3° dH, about 4° dH, about 5° dH, about 6° dH, about7° dH, about 8° dH, about 9° dH, about 10° dH, about 11° dH, about 12°dH, about 13° dH, about 14° dH, about 15° dH, about 16° dH, about 17°dH, about 18° dH, about 19° dH, about 20° dH, about 21° dH, about 22°dH, about 23° dH, about 24° dH, about 25° dH, about 26° dH, about 27°dH, about 28° dH, about 29° dH, about 30° dH. Under typical Europeanwash conditions, the degree of hardness is about 16° dH, under typicalUS wash conditions about 6° dH, and under typical Asian wash conditions,about 3° dH.

The present invention relates to a method of cleaning a fabric, adishware or hard surface with a detergent composition comprising aprotease variant of the invention.

A preferred embodiment concerns a method of cleaning, said methodcomprising the steps of: contacting an object with a cleaningcomposition comprising a protease variant of the invention underconditions suitable for cleaning said object. In a preferred embodimentthe cleaning composition is a detergent composition and the process is alaundry or a dish wash process.

Still another embodiment relates to a method for removing stains fromfabric which comprises contacting said a fabric with a compositioncomprising a protease of the invention under conditions suitable forcleaning said object.

In a preferred embodiment, the compositions for use in the methods abovefurther comprises at least one additional enzyme as set forth in the“other enzymes” section above, such as an enzyme selected from the groupof hydrolases such as proteases, lipases and cutinases, carbohydrasessuch as amylases, cellulases, hemicellulases, xylanases, and pectinaseor a combination hereof. In yet another preferred embodiment thecompositions for use in the methods above comprises a reduced amount ofat least one or more of the following components a surfactant, abuilder, a chelator or chelating agent, bleach system or bleachcomponent or a polymer.

Also contemplated are compositions and methods of treating fabrics(e.g., to desize a textile) using one or more of the protease of theinvention. The protease can be used in any fabric-treating method whichis well known in the art (see, e.g., U.S. Pat. No. 6,077,316). Forexample, in one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a protease in asolution. In one aspect, the fabric is treated with the solution underpressure.

In one embodiment, the protease variant is applied during or after theweaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The protease variant can be applied to remove thesesizing protein or protein derivatives. After the textiles have beenwoven, a fabric can proceed to a desizing stage. This can be followed byone or more additional fabric processing steps. Desizing is the act ofremoving size from textiles. After weaving, the size coating should beremoved before further processing the fabric in order to ensure ahomogeneous and wash-proof result. Also provided is a method of desizingcomprising enzymatic hydrolysis of the size by the action of an enzyme.

All issues, subject matter and embodiments which are disclosed forprotease variants in this application are also applicable for methodsand uses described herein. Therefore, it is explicitly referred to saiddisclosure for the methods and uses described herein as well.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods

Automatic Mechanical Stress Assay (AMSA) for Laundry

In order to assess the wash performance in laundry washing experimentsmay be performed, using the Automatic Mechanical Stress Assay (AMSA).With the AMSA, the wash performance of a large quantity of small volumeenzyme-detergent solutions can be examined. The AMSA plate has a numberof slots for test solutions and a lid firmly squeezing the laundrysample, the textile to be washed against all the slot openings. Duringthe washing time, the plate, test solutions, textile and lid arevigorously shaken to bring the test solution in contact with the textileand apply mechanical stress in a regular, periodic oscillating manner.For further description see WO02/42740 especially the paragraph “Specialmethod embodiments” at page 23-24.

The wash performance is measured as the brightness of the colour of thetextile washed. Brightness can also be expressed as the intensity of thelight reflected from the sample when illuminated with white light. Whenthe sample is stained the intensity of the reflected light is lower,than that of a clean sample. Therefore the intensity of the reflectedlight can be used to measure wash performance.

Colour measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak, Midtager 29, DK-2605 Brondby, Denmark), which is used tocapture an image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image are converted into values for red,green and blue (RGB). The intensity value (Int) is calculated by addingthe RGB values together as vectors and then taking the length of theresulting vector:Int+√{square root over (r ² +g ² +b ²)}.

TABLE 1a Composition of model detergents and test materials LaundryModel detergent and test materials are as follows: Laundry powder Sodiumcitrate dihydrate 32.3% model detergent A Sodium-LAS 24.2% Sodium laurylsulfate 32.2% Neodol 25-7 (alcohol ethoxylate) 6.4% Sodium sulfate 4.9%Laundry liquid Water 30.63% model detergent B Sodium hydroxide 2.95%Dodecylbenzensulfonic acid 11.52% Fatty acids (Soya) 5.50%Propane-1,2-diol (MPG) 5.05% Water 17.38% C13-alcohol ethoxylate, 10.50%Diethylenetriaminepentakis (methylenephosphonic acid) (DTMPA) 3.08%Triethanolamine (TEA) 2.22% Fatty acids (Coco) 4.50% Sodium citratemonohydrate 1.00% Ethanol 4.63% Syntran 5909 (opacifier) 0.30% Perfume0.35% Test material PC-03 (Chocolate-milk/ink on cotton/polyester) C-05(Blood/milk/ink on cotton)

TABLE 1b Liquid model detergent for stability test Liquid 0.3 to 0.5%xanthan gum, model detergent 0.2 to 0.4% antifoaming agent, 6 to 7%glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAEOS (fatty alcohol ethersulfate), 24 to 28% nonionic surfactants, 1% boric acid, 1 to 2% sodiumcitrate (dihydrate), 2 to 4% soda, 14 to 16% coconut fatty acid, 0.5%HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP(polyvinylpyrrolidone), 0 to 0.05% optical brighteners, 0 to 0.001% dye,remainder deionized water.General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Protease Activity Assays:

1) Suc-AAPF-pNA Activity Assay:

The proteolytic activity can be determined by a method employing theSuc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-N itroanilide, and itis a blocked peptide which can be cleaved by endo-proteases. Followingcleavage a free PNA molecule is liberated and it has a yellow colour andthus can be measured by visible spectrophotometry at wavelength 405 nm.The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400,dissolved in DMSO).

The protease sample to be analyzed was diluted in residual activitybuffer (100 mM Tris pH8.6). The assay was performed by transferring 60μl of diluted enzyme samples to 96 well microtiter plate and adding 140μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). Thesolution was mixed at room temperature and absorption is measured every20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the protease in question under the given set of conditions.The protease sample should be diluted to a level where the slope islinear.

Example 1: Preparation and Testing of Protease Variants

Preparation and Expression of Variants

Site-directed variants were constructed of the TY145 protease (SEQ IDNO: 3) comprising specific insertions/deletions/substitutions in the 170to 180 region on the N-terminal side according to the invention. Thevariants were made by traditional cloning of DNA fragments (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, 1989) using PCR together with properly designed mutagenicoligonucleotides that introduced the desired mutations in the resultingsequence. Mutagenic oligos were synthesized corresponding to the DNAsequence flanking the desired site(s) of mutation, separated by the DNAbase pairs defining the insertions/deletions/substitutions. In thismanner, the variants listed in table 2a below were constructed andproduced.

Fermentation of Variants

Fermentation may be performed by methods well known in the art or asfollows. A B. subtilis strain harboring the relevant expression plasmidwas streaked on a LB-agar plate with a relevant antibiotic (6 μg/mlchloramphenicol), and grown overnight at 37° C.

The colonies were transferred to 100 ml PS-1 media supplemented with therelevant antibiotic in a 500 ml shaking flask containing a rich media(e.g. PS-1: 100 g/L Sucrose (Danisco cat. no. 109-0429), 40 g/L crustsoy (soy bean flour), 10 g/L Na₂HPO₄.12H₂O (Merck cat. no. 6579), 0.1ml/L Pluronic PE 6100 (BASF 102-3098)). Cultivation typically takes 4days at 30° C. shaking with 220 rpm. Cells and other undissolvedmaterial were removed from the fermentation broth by centrifugation at4500 rpm for 20-25 minutes. Afterwards the supernatant was filtered toobtain a clear solution.

Example 2

In this example, the above-described PNA-Suc-AAPF assay is used todetermine the residual protease activity after incubation in thepresence of liquid model detergent. In general the residual proteaseactivity was determined after incubation in liquid model detergent(table 1b) (final concentration of 90%) at the indicated temperaturesand incubation times and the activity is then compared to the activityof a unstressed incubated at 4° C. For the determination of proteasestability in detergent the enzymes to be tested were adjusted to aconcentration of 0.15 mg/ml of enzyme protein by dilution in enzymedilution buffer (100 mM Tris pH 8.6, 0.0225% (w/V) Brij-35, 2 mM CaCl₂).30 μl of the protease solution and 270 μl liquid detergent (liquid modeldetergent, table 1b) was transferred to a 96 well microtiter plate (Nunc96U PP) in 4 replicates. One small magnet (5×2 mm) was placed in eachwell, and the blend was mixed for 30 minutes at room temperature on amagnetic stirrer. After mixing 20 μl is transferred to new 96 wellmicrotiter plate and incubated at 4° C. for 24 hours (unstressedsample). Heat seal with alu-foil carefully microtiter plate and incubateat indicated temperature for 24 hours (stressed samples). Afterincubation, the samples on the plates were analyzed for proteaseactivity as described in the PNA-Suc-AAPF Assay for determination ofresidual protease activity. It should be noted, that in order to reduceinterference from other detergent ingredients than the enzyme on theassay, both unstressed and stressed samples were diluted to the sameprotein concentration.

After incubation, withdraw 20 μl of stressed samples and add 150 μlresidual activity buffer (100 mM Tris pH8.6), and mix using magneticstirrer for 5 minutes. Transfer 60 μl of diluted sample to new 96 wellmicrotiter plate. Before use prepare PNA-Suc-AAPF substrate workingsolution in residual activity buffer (0.72 mg/ml in 100 mM Tris pH8.6).Add 140 μl substrate working solution to diluted sample, mix and measureimmediately absorbance at 405 nm for 5-10 minutes every 20 seconds atroom temperature. Use Vmax only from linear range of kinetic curves.Repeat residual activity measurement for unstressed sample by adding 150μl residual activity buffer (100 mM Tris pH8.6) to microtiter plate with20 μl unstressed sample (incubated at 4° C.), and mix using magneticstirrer for 5 minutes. Transfer 60 μl of diluted sample to new 96 wellmicrotiter plate. Add 140 μl substrate working solution to dilutedsample, mix and measure immediately absorbance at 405 nm for 5-10minutes every 20 seconds at room temperature. Use Vmax only from linearrange of kinetic curves.

It was ensured in all experiments that the reference protease wasincluded at least once on all test microtiter plates.

The residual activity (% RA) was calculated as % RA=100*Vmax (stressedsample)/Vmax (unstressed sample).

The half-life (T½(h)) is calculated: T½ (hours)=T (hours)*LN(0.5)/LN(%RA/100) with T being incubation time (hours) and % RA is residualactivity.

TABLE 3 stability of variants measured at 35° C. TY-145 (SEQ ID NO 3) 18V124G 20 I121S 31 I121C 32 I121V 27 I121T 33 V162W 27 V162R 34 V124A 19I137E 60 I137C 27 I137S 28 I137A 34 I137V 16 I137M 26 I137T 23 I137Q 42I137G 28 V124A S133T 21 V124A R130A 25 I121V S175P 96 I137E S144Q 71I137E S144R 58 I137M S144Q 38

TABLE 4 Stability of variants measured at 42° C. TY-145 (SEQ ID NO 3) 0I121V S175P 7 I137E S175P 7 I121V S175A 3 I137E S175A 4 I121T S175P 8I137E S173Y G174S S175A F180Y 11 I121V S173P G174K S175P N176G T177SF180Y 52 I137E S173P G174K S175P N176G T177S F180Y 41

TABLE 5 Stability of variants measured at 47° C. TY-145 (SEQ ID NO 3) 0I137E 6 I121V S175P 6 I137E S175P 6 I121V S175A 6 I137E S175A 6 I121TS175P 6 I137M S173P 30 I121V S144Q 6 T74M I137E S173P 26 L34I I137ES173P 36 Y39D I137E S173P 39 T40P I137E S173P 46 I137E S173P I247M 41I137E S173P H256F 31 I137E S173Y S175A F180Y 6 V162R S173P S175P F180Y96 I137E S173Y G174S S175A F180Y 6 V162R S173Y G174S S175A F180Y 6 I121VS173Y G174S S175A F180Y 6 I137E S173P S175V T177S F180Y 34 I137E S173YG174S S175P F180Y 7 V162R S173P G174T S175V T177S F180Y 74 I121V S173PG174T S175V T177S F180Y 49 I121V I137E S173Y G174S S175A F180Y 6 L81VI137E S173Y G174S S175A F180Y 6 I137E S173Y G174S S175A F180Y T241P 6Q70N I137E S173Y G174S S175A F180Y 6 I137E S173Y G174S S175A F180Y S274I6 I137E S173Y G174S S175A F180Y T297P 8 I137E S144Q S173Y G174S S175AF180Y 6 T40L I137E S173Y G174S S175A F180Y 6 I137E S173P S175P N176GT177S F180Y 6 I137E S173Y G174S S175A F180Y V286Q 6 V162R S173P G174KS175P N176G T177S F180Y 11 I121V S173P G174K S175P N176G T177S F180Y 8I137E S173P G174K S175P N176G T177S F180Y 9 I121V I137E S173P G174TS175V T177S F180Y 73 I137E S173P G174T S175V T177S F180Y T241P 90 I137ES173P G174T S175V T177S F180Y V286Q 75 I137E S173P G174T S175V T177SF180Y T297P 96 I137E S171N S173P G174T S175V T177S F180Y 74 I137E S173PG174K S175A N176G T177S F180Y 6 I137E S173P G174T S175V T177S F180YS274I 64 I137E S144Q S173P G174T S175V T177S F180Y 56 I121V I137E S173PG174K S175P N176G T177S F180Y 13 Q70N I137E S173P G174K S175P N176GT177S F180Y 13 I137E S173P G174K S175P N176G T177S F180Y S274I 12 I137ES173P G174K S175P N176G T177S F180Y V286Q 12 I137E S173P G174K S175PN176G T177S F180Y T297P 23 I137E S171N S173P G174K S175P N176G T177SF180Y 15 I137E S144Q S173P G174K S175P N176G T177S F180Y 11 V162R S171NS173P G174K S175P N176G T177S F180Y 16 I137E S171N S173P G174K S175PN176G T177S F180Y T241P 22 I137E S171N S173P G174K S175P N176G T177SF180Y T297P 41

TABLE 6 Stability of variants measured at 52° C. TY-145 (SEQ ID NO 3) 0T40P I137E S173P 7 I137E S173P S175P F180Y 39 V162R S173P S175P F180Y 45V162R S173P G174T S175V T177S F180Y 16 I137E S173P G174T S175A T177SF180Y 14 I137E S173P G174T S175V T177S F180Y V286Q 18 I137E S171N S173PG174T S175V T177S F180Y 23 I137E S144Q S173P G174T S175V T177S F180Y 15I137E S173P G174T S175P T177S F180Y T297P 96

The invention claimed is:
 1. A protease variant, comprising asubstitution in the hydrophobic cluster corresponding to position 137 ofSEQ ID NO: 3, wherein (a) the variant has a sequence identity to SEQ IDNO: 3 of at least 85% and less than 100%, and (b) the variant hasprotease activity.
 2. The variant according to claim 1, which comprisesa substitution at two or more positions corresponding to position 137and any of the positions 121, 124, or
 162. 3. The variant according toclaim 2, wherein the amino acid at the position corresponding toposition 124 of SEQ ID NO: 3 is Ala.
 4. The variant according to claim1, wherein the amino acid at the position corresponding to position 137of SEQ ID NO: 3 is selected from the group consisting of Glu, Cys, Ser,Ala, Met, Thr, Gln, and Gly.
 5. The variant according to claim 2,wherein the amino acid at the position corresponding to position 162 ofSEQ ID NO: 3 is selected from the group consisting of Trp and Arg. 6.The variant of claim 2, wherein the amino acid at the positioncorresponding to position 121 of SEQ ID NO: 3 is selected from the groupconsisting of Ser, Cys, Val and Thr.
 7. The variant of claim 1, whichfurther comprises one or more substitutions selected from the groupconsisting of L34I, Y39D; T40{D,P,L}; Q70N; T74M; L81{F,H,V}; A102T;R130A, G132 {I,E}; S133T S144{Q,R}; D155N; G159S; V162R, S171 {W, K, E,N}; S173{P,V}; G174{S,T}; S175 {A, V, P}; N176G; T177S; G179 {C, V, Q,S, T, E, H, K, M, N, A, Y}; F180Y; T241P; I247M; H256F, S274I; V286Q andT297P.
 8. The variant of claim 2, comprising any of the followingsubstitutions: I137M S173P I121V S175P I137E S175P I121V S175A I137ES175A I137E S144Q I137E S144R I137M S144R I121T S175P I137M S144Q V124AS133T V124A R130A I137E S173Y G174S S175A F180Y V162R S173P G174T S175VT177S F180Y I121V S173P G174T S175V T177S F180Y V162R S173P G174K S175PN176G T177S F180Y I121V S173P G174K S175P N176G T177S F180Y I137E S173PG174K S175P N176G T177S F180Y I121V I137E S173Y G174S S175A F180Y L81VI137E S173Y G174S S175A F180Y I137E S173Y G174S S175A F180Y T241P Q70NI137E S173Y G174S S175A F180Y I137E S173Y G174S S175A F180Y S274I I137ES173Y G174S S175A F180Y T297P I137E S173P G174T S175V T177S F180Y T241PI137E S173P G174T S175V T177S F180Y V286Q I137E S171N S173P G174T S175VT177S F180Y I137E S173P G174T S175A T177S F180Y I121V I137E S173P G174KS175P N176G T177S F180Y Q70N I137E S173P G174K S175P N176G T177S F180YI137E S173P G174K S175P N176G T177S F180Y S274I I137E S173P G174K S175PN176G T177S F180Y V286Q I137E S173P G174K S175P N176G T177S F180Y T297PI137E S171N S173P G174K S175P N176GT177S F180Y I137E S173P G174K S175AN176G T177S F180Y V162R S173Y G174S S175A F180Y I121V S173Y G174S S175AF180Y I121V S144Q V162R S173P G174T S175V T177S F180Y L81V I137E S173YG174S S175A F180Y I121V I137E S173P G174T S175V T177S F180Y I137E S173PG174T S175V T177S F180Y T297P I137E S171N S173P G174T S175V T177S F180YV162R S173Y G174S S175A F180Y I137E S144Q S173Y G174S S175A F180Y T40LI137E S173Y G174S S175A F180Y I137E S173Y S175A F180Y I137E S173P S175PN176G T177S F180Y I137E S173Y G174S S175A F180Y V286Q I137E S173P G174TS175V T177S F180Y S274I T74M I137E S173P L34I I137E S173P Y39D I137ES173P T40P I137E S173P I137E S173P I247M I137E S173P H256F I137E S144QS173P G174T S175V T177S F180Y I137E S144Q S173P G174K S175P N176G T177SF180Y I137E S173Y G174S S175P F180Y V162R S171N S173P G174K S175P N176GT177S F180Y I137E S171N S173P G174K S175P N176G T177S F180Y T241P I137ES173P S175P F180Y V162R S173P S175P F180Y I137E S173P S175V T177S F180Y.


9. The variant of claim 1, which has an improved detergent stabilitycompared to the protease with SEQ ID NO:
 3. 10. The variant of claim 1,wherein the variant is selected from the group consisting of: a. apolypeptide having at least 85% sequence identity to the maturepolypeptide of SEQ ID NO: 2; b. a polypeptide encoded by apolynucleotide having at least 85% identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or a sequence encoding the maturepolypeptide of SEQ ID NO: 2; and c. a fragment of the mature polypeptideof SEQ ID NO: 2, which has protease activity.
 11. The variant of claim1, wherein the variant has at least 90% but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO:
 3. 12. The variant ofclaim 1, wherein the total number of alterations compared to SEQ ID NO:3 is 1-20.
 13. A method for obtaining a protease variant, comprisingintroducing into a parent subtilase a substitution at a positioncorresponding to position 137 of SEQ ID NO: 3, wherein the variant hasan amino acid sequence which is at least 85% identical to SEQ ID NO: 3,and recovering the variant.
 14. A cleaning composition comprising avariant according to claim
 1. 15. A method for removing a stain from asurface, the method comprising contacting the surface with a compositionaccording to claim
 14. 16. The variant of claim 1, wherein the varianthas at least 95% but less than 100%, sequence identity to the maturepolypeptide of SEQ ID NO:
 3. 17. The variant of claim 1, wherein thevariant has at least 97% but less than 100%, sequence identity to themature polypeptide of SEQ ID NO:
 3. 18. The variant of claim 10, whereinthe variant is a polypeptide having at least 90% sequence identity tothe mature polypeptide of SEQ ID NO:
 2. 19. The variant of claim 10,wherein the variant is a polypeptide having at least 95% sequenceidentity to the mature polypeptide of SEQ ID NO:
 2. 20. The method ofclaim 13, further comprising introducing into the parent subtilase asubstitution at any one or more position corresponding to position 121,124, or 162 of SEQ ID NO: 3.